White, brown, cave…

Ancient Bears: Read from the Genome book

Alyona Litvinenko, "Science in Siberia" Photos provided by Anna Druzhkova

A few million years ago there were no white, brown, or Himalayan bears, but thanks to evolution and climate changes, the ancestors of clubfoot adapted to environmental conditions, dividing into species already known to us. The analysis of ancient DNA, which is carried out by scientists from the Institute of Molecular and Cellular Biology SB RAS, helps to find out how long certain representatives of this family have existed and how closely related they are.

Ancient DNA is obtained from any archaeological or paleontological samples: bone remains, teeth, mummified tissue, wool, feathers, shells, plant grains. Scientists are assisted in this by the so–called polymerase chain reaction, an experimental method that makes it possible to increase small concentrations of DNA fragments in biological material. 

When it became possible to isolate DNA from ancient samples, sciences began to be created at the junction of different disciplines – including archaeogenetics (or paleogenetics), which includes the analysis of ancient DNA, where molecular methods are used for a new look at archaeological samples. 

For the first time, ancient DNA was isolated in 1984 from a museum exhibit of the quaggi: an extinct species of the equine family. At first, the methods were ineffective, but with the development of PCR, mass studies of DNA from ancient samples, including extinct mammals, dinosaurs and even organisms encased in amber, began. The scientific world was optimistic about such work, but with the use of PCR there was such a problem as contamination – accidental contamination of an object with foreign DNA.

– For example, dinosaurs eventually turned out to be the closest relatives of chickens – although, most likely, someone from the laboratory ate a dish with such meat that day, - says Anna Sergeevna Druzhkova, a junior researcher at the Laboratory of Comparative Genomics of the IBC SB RAS. – Such errors are due to the fact that the polymerase chain reaction goes better on DNA from modern samples – after all, you can take blood or muscle tissue from them, and they have not been exposed to destructive factors for a long time. 

If you have special equipment and methods, the genome of various animals is read as an open book, but ancient samples are often torn books or scraps of pages with texts that have not been preserved. Sometimes the DNA is simply not preserved in the sample – there remains only a dump of what was nearby: information about the soil, people who dug up the sample, microorganisms that took part in the decomposition, various fungi and bacteria. Under ideal conditions, the calculation of DNA preservation is one million years, but not every sample after death gets into the "refrigerator" at -74 °C. Therefore, when work with DNA began, scientists first of all paid attention to samples from permafrost (for example, mammoths) or from caves with a dry microclimate, thanks to which the body was mummified.

– In our studies, there were bone samples that underwent passage through the digestive tract of hyenas, which also affected DNA, – adds Anna Druzhkova. – In order to distinguish whether you isolated DNA from the sample you were looking for, a scientist sitting next to you, or a dog that rubbed against his leg at home, strict authenticity criteria were invented. They mean working in laboratories in a special spacesuit, treating all surfaces with special decontaminators and changing gloves every time they come into contact with a new sample.

For a more accurate result, when preparing genomic libraries, it is necessary to remove contamination by excluding high-molecular fragments, since they belong to modern organisms. Ancient DNA degrades and can contain from 70 to 120 pairs of nucleotides, while in modern DNA there are about 1000 – even if the smallest fragments are taken into account. Therefore, it is safe to exclude large ones, not to mention the fact that greater coverage during sequencing makes it possible to select the necessary sequences in controversial moments that scientists consider to be contamination.

– When we started our work with ancient DNA, the question arose where to take samples, and here the Institute of Archaeology and Ethnography SB RAS helped us, – adds the researcher. – It has collections of bones of various mammals, with the help of which it is possible to reconstruct the evolutionary history of a species. For our study, samples of brown bears and extinct small cave bears were taken. There was a lot of controversy about the latter: other geneticists have already examined bone samples from Altai and claimed that only brown bears lived there. However, based on morphometric indicators, our colleagues believed that other species lived in Altai. 

Some of the samples were taken from Denisova Cave, a place where DNA is well preserved due to the microclimate. Bones washed up on the banks of the Siberian rivers Chumysh and Chik were also used. The age of the samples from there was approximately 32,000 years, and despite the fact that the bones were blackened and poorly preserved, modern methods have made it possible to obtain a mitochondrial genome even from them.

– To isolate DNA, we use a widespread method, – explains Anna Druzhkova. – To begin with, we remove the upper layer of the bone (where there are most contaminants) with a diamond disc, grind a small fragment into powder and dissolve it in a special solution, cleaning it from contaminants that interfere with PCR. Naturally, everything that was nearby stands out from the ancient sample: fungi, bacteria, people.

The content of mitochondrial DNA in the sample is small, since nuclear DNA and various contaminants occupy a certain place. Therefore, scientists use the enrichment protocol using probes (fragments) from the DNA of a modern sample of the same or closely related species. The most similar fragments of ancient DNA, containing unique bar codes, are attracted to modern DNA like a magnet - like identical tags, according to which, during bioinformatic analysis, all sequences from an ancient sample can be sorted and not confused with a modern one taken for enrichment. Thus, from the entire mixture of DNA from an ancient sample, scientists "pull out" the desired mitochondrial fragments.

– The brown bears that appeared in our study really belong to the brown ones, without errors, – the researcher emphasizes. "It's the same with the cavemen. This is an important result, because it is molecularly proven that representatives of different species from the bear family lived on the territory of Western Siberia.

Thanks to the purification from contamination and the enrichment of libraries for sequencing, scientists were able to collect mitochondrial genomes and build a phylogenetic tree reflecting the evolutionary relationships between different representatives of the bear. The fact is that as a result of the last glacial maximum, a small group of brown bears settled throughout Eurasia. Researchers from the IMKB SB RAS managed to show that representatives from earlier migration waves lived on the territory of Siberia, traces of which remained in modern bear populations of the Japanese Islands and Alaska. Thus, the Altai bears from the Kolyvan excavation, 3,500 years old, and from the Denisova cave, 5,000 years old, are part of the final wave of migrants who settled in the last 10,000 years, and the bear from the Chumysh River, which is 32,000 years old, belongs to the penultimate migration wave that took place 30-50 thousand years ago.

– We also had a bear about 32,000 years old from the Chik River, – says Anna Druzhkova. – He also belongs to the group of brown bears, the closest to modern white and brown bears, which are now found on the Admiralteysky and Baranov Islands. When we realized that there was not enough data on modern bears in Siberia, since the territory is very large, colleagues shared with us a modern sample from Irkutsk. We made an enrichment on it, and revealed that it also belongs to this unique group from the penultimate migration wave – one of forty modern bears turned out to have such a unique haplotype!

According to previously obtained calculations, the common ancestor with brown and cave bears lived about three million years ago. Based on the research of scientists from the IMKB SB RAS, it can be argued that the brown bears that existed in Siberia were very diverse, and their traces can really be found throughout the modern area of fleecing.

American and Tibetan bears were the very first to separate from the common ancestor, and then cave bears, white bears, and then all brown bears. Many transformations were influenced by climatic changes and ice ages, so the habitats of brown and white were mixed. However, the whites were a separate species about 700,000 years ago according to theoretical calculations based on the analysis of the nuclear genes of modern bears.

Siberian scientists used grizzly and Himalayan bear as an out-group (the most closely related species), but they are on a far-lying branch. According to mitochondrial DNA, brown bears are also related to whites, but if you look at the nuclear one, it becomes clear that whites are definitely an earlier separated branch. Therefore, it may be the same with cave bears, because mitochondrial DNA provides information only on the maternal side. It is not yet possible to say that small cave bears are a separate species among cave bears, but it can be said for sure that they really differ from brown bears.

Portal "Eternal youth" http://vechnayamolodost.ru  06.07.2017